Natural killer cells

Abstract

This invention relates to expanded Natural Killer (NK) cell populations, to methods of producing the same and therapeutic applications thereof. More specifically, the invention relates to increasing the number of CD16+ NK cells within expanded NK cell populations without the need for exogenous gene expression.

1 . An ex vivo method for producing an expanded population of CD16 +  Natural Killer (NK) cells, comprising the steps of:
 a) culturing an haematopoietic progenitor cell (HPC) comprising sample obtained from an individual in medium which does not induce differentiation of the HPCs for between about 2 to about 8 days to produce a pre-differentiation HPC population; and 
 b) culturing the pre-differentiation HPC population in medium which induces differentiation of the HPCs to NK. 
 
     
     
         2 . An ex vivo method for increasing the number of CD16 +  NK cells in an expanded NK cell population, comprising the steps of:
 a) culturing an haematopoietic progenitor cell (HPC) comprising sample obtained from an individual in medium which does not induce differentiation of the HPCs for between about 2 to about 8 days to produce a pre-differentiation HPC population; and 
 b) culturing the pre-differentiation HPC population in medium which induces differentiation of the HPCs to NK. 
 
     
     
         3 . A method according to  claim 1 or 2 , wherein in step (a) the HPCs are cultured for between about 2 days to about 6 days, optionally for between about 4 days to about 6 days. 
     
     
         4 . A method according to  any one of the preceding claims , wherein the HPCs are cultured in the presence of a Notch ligand for at least part of step (a). 
     
     
         5 . A method according to  claim 4 , wherein:
 (i) the HPCs are cultured in a vessel that is coated with the Notch ligand for at least part of step (a); and/or   (ii) the Notch ligand is delta-like ligand 4 (DLL4), or a fragment thereof which retains the function of DLL4;   wherein preferably in step (a) the HPCs are cultured in the absence of the Notch ligand for about 1 day followed by culture in the presence of the Notch ligand for the remainder of step (a).   
     
     
         6 . A method according to  any one of the preceding claims , wherein:
 (i) in step (a) the HPCs are cultured in the presence of a compound which inhibits the action of REV-ERB; and/or   (ii) in step (b) the pre-differentiation HPC population is cultured in the presence of a compound which inhibits the action of REV-ERB.   
     
     
         7 . A method according to  claim 6 , wherein said compound:
 (i) increases E4bp4 expression by decreasing REV-ERB activity;   (ii) decreases the activity of REV-ERB-α and/or REV-ERB-β, preferably REV-ERB-β;   (iii) decreases the activity of REV-ERB-α and REV-ERB-β;   (iv) is a REV-ERB antagonist, preferably an antagonist of REV-ERB-α and REV-ERB-β;   (v) is selected from a small molecule, a PROTAC reagent, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a micro RNA, an antisense RNA, an aptamer, an antibody, a ribozyme, a peptide or a peptidomimetic, preferably a small molecule; and/or   (vi) is SR8278,   
       
         
           
           
               
               
           
         
          or GSK1362. 
       
     
     
         8 . A method according to  any one of the preceding claims , wherein the medium which does not induces differentiation of the HPCs in step (a) and/or the medium which induces differentiation of the HPCs to NK in step (b) does not comprise IL-3, preferably wherein the medium which induces differentiation of the HPCs to NK in step (b) does not comprise IL-3. 
     
     
         9 . A method according to  any one of the preceding claims , wherein:
 (i) the medium in step (a) comprises at least one of Flt3L, GM-CSF, IL-3, IL-6, TPO and/or stem cell factor (SCF), preferably Flt3L, GM-CSF, IL-3, IL-6, TPO and SCF; and/or   (ii) the medium in step (b) comprises IL-7, Flt3L, IL-15, and/or SCF, preferably IL-7, Flt3L, IL-15 and SCF.   
     
     
         10 . A method according to  any one of the preceding claims , wherein the step (a) and/or step (b) is carried out in the absence of a stromal support cell, preferably wherein both step (a) and step (b) are carried out in the absence of a stromal support cell. 
     
     
         11 . A method according to  any one of the preceding claims , wherein the sample of HPCs is obtained from bone marrow, cord blood and/or peripheral blood. 
     
     
         12 . A method according to  any one of the preceding claims , wherein the proportion of CD16 +  NK cells is increased compared with the proportion of CD16 +  NK cells produced by a corresponding method in which step (a) is omitted. 
     
     
         13 . A method according to  any one of the preceding claims , wherein the expanded NK cell population comprises at least 10% CD16 +  NK cells, preferably at least 15% CD16 +  NK cells, more preferably at least 20% CD16 +  NK cells, even more preferably at least 30% CD16 +  NK cells. 
     
     
         14 . A method according to  any one of the preceding claims , wherein the expanded NK cell population exhibits at least 30% greater antibody-dependent cellular cytotoxicity (ADCC), preferably at least 50% greater ADCC, compared with NK cells produced by a corresponding method in which step (a) is omitted. 
     
     
         15 . A method according to  any one of the preceding claims , wherein which does not comprise a further step of introducing exogenous nucleic acid into the HPCs and/or NK cells. 
     
     
         16 . An expanded population of CD16 +  NK cells, wherein at least 10% of the NK cells are CD16 +  NK cells, preferably at least 15% of the NK cells are CD16 +  NK cells, more preferably at least 20% of the NK cells are CD16 +  NK cells, even more preferably at least 30% of the NK cells are CD16 +  NK cells. 
     
     
         17 . An expanded population of CD16 +  NK cells obtained by the method of any one of  claims 1 to 15 , wherein at least 10% of the NK cells are CD16 +  NK cells, preferably at least 15% of the NK cells are CD16 +  NK cells, more preferably at least 20% of the NK cells are CD16 +  NK cells, even more preferably at least 30% of the NK cells are CD16 +  NK cells. 
     
     
         18 . An expanded population of CD16 +  NK cells according to  claim 16 or 17 , wherein the CD16 +  NK cells do not comprise exogenous nucleic acid. 
     
     
         19 . An expanded population of CD16 +  NK cells according to any one of  claims 16 to 18 , wherein the expanded NK cell population exhibits at least 30% greater ADCC, preferably at least 50% greater ADCC, more preferably at least 70% greater ADCC, compared with NK cells produced by a corresponding method in which step (a) is omitted. 
     
     
         20 . A composition comprising an expanded NK cell population as defined in any one of  claims 16 to 19  and a pharmaceutically acceptable carrier, diluent and/or excipient. 
     
     
         21 . An expanded population of CD16 +  NK cells as defined in any one of  claims 16 to 19  or a composition as defined in  claim 20  for use in a method of therapy. 
     
     
         22 . An expanded population of CD16 +  NK cells or composition for use according to  claim 21 , wherein said method of therapy is a method of treating a disease or disorder selected from cancer, an infectious disease (acute or chronic), an autoimmune disease or a disease or disorder related to female infertility or pregnancy. 
     
     
         23 . An expanded population of CD16 +  NK cells or composition for use according to  claim 21 or 22 , wherein said method of therapy is a method of treatment of a viral infection, a bacterial infection, a protist infection, a fungal infection and/or a helminth infection. 
     
     
         24 . An expanded population of CD16 +  NK cells or composition for use according to any one of  claims 21 to 23 , which is used in combination with antibody-mediated immunotherapy. 
     
     
         25 . An expanded population of CD16 +  NK cells or composition for use according to  claim 24 , wherein said expanded population of CD16 +  NK cells or composition is for administration before, simultaneously with, or after administration of the antibody-mediated immunotherapy.